Report of two distinct ribotypes in ITS sequences of Phalaris arundinacea (Poaceae) in western Canada and Alaska

Biodiversity Data Journal 11: e101257 CO)
doi: 10.3897/BDJ.11.e101257 open access
Data Paper

Report of two distinct ribotypes in ITS sequences
of Phalaris arundinacea (Poaceae) in western
Canada and Alaska

Diana M. Percy*, Quentin C. B. Cronk?

+ Department of Botany, University of British Columbia, Vancouver, Canada
§ Beaty Biodiversity Museum, University of British Columbia, Vancouver, Canada

Corresponding author: Diana M. Percy (diana.percy@ubc.ca)

Academic editor: Marcin Nobis

Received: 31 Jan 2023 | Accepted: 28 Mar 2023 | Published: 11 Apr 2023

Citation: Percy DM, Cronk QCB (2023) Report of two distinct ribotypes in ITS sequences of Phalaris
arundinacea (Poaceae) in western Canada and Alaska. Biodiversity Data Journal 11: e101257.
https://doi.org/10.3897/BDJ.11.e101257

Abstract

Background

Phalaris arundinacea L. (reed canary grass) is a widely occurring grass throughout the
Northern Hemisphere. In North America, it is thought to consist of introduced agricultural
forms from Europe as well as native populations.

New information

During a survey of Phalaris arundinacea in western Canada, we discovered two distinct
ribotypes in the sequences of the internal transcribed spacer (ITS) of the nuclear ribosomal
DNA: one full length (ITS-long) and one with a seven base pair deletion (ITS-short). In
addition, ITS-long plants have fixed heterozygosity indicating possible polyploidy.
Phylogenetic analysis reveals that ITS-short is a unique ribotype that characterises an
intraspecific clade. We designed an efficient PCR-based assay that allows sizing of a

© Percy D, Cronk Q. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC
BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are
credited.

2 Percy D, Cronk Q

238/245 base pair fragment in a capillary sequencer. This approach provides a novel
marker that could be useful in future surveys of Phalaris arundinacea.

Keywords

internal transcribed spacer, invasive plant, Phalaris, reed canary grass

Introduction

Phalaris arundinacea L., commonly called reed canary grass (RCG), is a Eurasian and
North American perennial grass, with many uses in agriculture (Jakubowski et al. 2011)
and biomass energy (Lewandowski et al. 2003). In North America, native populations are
considered under threat from invasion and replacement by vigorous introduced genotypes
of P arundinacea that have now become a significant invader of wetland and riparian
habitats in North America (Lavergne and Molofsky 2004) with considerable ecological
impacts (Spyreas et al. 2010). The distribution of Phalaris arundinacea in North America,
based on databased herbarium specimens, is shown in Fig. 1.

Figure 1. EES

Map of North American Phalaris arundinacea herbarium specimens from the Global
Biodiversity Information Facility (GBIF; accessed October 2021). Red = 1822-1940; blue =
1941-2018. The dotted line marks the boundary of the western cordilleras.

Molecular methods have often been used to distinguish populations of RCG, including
isozymes (Gifford et al. 2002), AFLP (Casler et al. 2009), SSR (Jakubowski et al. 2013,

Report of two distinct ribotypes in ITS sequences of Phalaris arundinacea ... 3

Jakubowski et al. 2014, Kettenring et al. 2019), ISSR (Anderson et al. 2016), DartSeq
(Noyszewski et al. 2019, Noyszewski et al. 2021) and ITS sequencing (Graper et al. 2021).
However, there is still much uncertainty and, in some cases, disagreement, regarding the
extent of distribution and location of present day native versus introduced RCG populations
in North America (Jakubowski et al. 2013). The aim of this note is to detail an easily scored
novel genetic marker that may be of use in future surveys of RCG.

Sampling methods
Description: Sources of material - herbarium and field

A total of 86 samples of Phalaris arundinacea were obtained from herbarium material and
additional targeted sampling carried out for this study (Tables 1, 2, Suppl. materials 1, 3).
Herbarium samples, from modern to 130 years old and in relatively good condition, were
selected for sampling from the University of British Columbia Herbarium (UBC) and the
Herbarium of the Bell Museum, University of Minnesota (MIN). Further dried leaf samples
(used in a previous study; Kettenring et al. (2019)) were kindly provided by Professor
Karen Mock of Utah State University. In addition, extensive field sampling was carried out
in Elk Island National Park, Alberta, where park authorities were concerned about the
ecologically harmful spread of, as well as appropriate control methods for, Phalaris
arundinacea. Further recent samples were sourced from Greater Vancouver. Voucher
specimens are deposited in UBC. As outgroups for the phylogenetic analyses, we used
eight individuals obtained from herbarium samples of P. aquatica Guss., P canariensis L.,
P. caroliniana Walter, P coerulescens Desf. and P. paradoxa L. (Suppl. material 2).

Table 1.

Herbarium specimens identified as ITS-short: determined by sequencing or sizing assay to have a
7 bp deletion in ITS2. An asterisk indicates one individual identified as ITS-short in assay data, but
putative hybrid in sequence data; and [] indicates the only sequence found on GenBank with the
ITS-short genotype. Region abbreviations: AB Alberta, AK Alaska, BC British Columbia, NWT
Northwest Territories.

Accession no. Herb. Date Locality Region Habitat

V100979 UBC 1950 Chilcotin, Madden Lake BC not recorded

V101113 UBC 1950 Chilcotin, Meldrum Creek BC marsh

*V152455 UBC 1974 near Shamrock, ca. 30 miles northwest BC in post-glacial bed of the Stuart
of Prince George River

V162493 UBC 1975 Beaver Lake, Wilson Creek Road, nr. BC swampy lake edge
Slocan Lake

V27934 UBC 1950 S. of Ft Smith NWT scattered in clumps along

dried-up slough

V67193 UBC 1957 Kootenay District, Flathead, Procter BC in 2 ft (60 cm) of water at lake
Lake edge

Accession no.

V67205
V67206
V88503

V111611

V242084

V119175

[KF753778]

V20064

Table 2.

Herb. Date

UBC 1957
UBC 1957
UBC 1958

UBC 1955

UBC 2014
ALA 1994

UBC 1945

Percy D, Cronk Q

Locality

Kootenay District, Sage Creek Lodge.
Flathead valley, Marl Lake

Kootenay, Nakusp, Wilson Lake.

5 mi (7.5 km) southeast of Fort
Simpson,

Yoho National Park, Hoodoo Creek
Campground area

Cook Inlet lowlands, Otter Creek at
Loop Road

Just east of Fort Saskatchewan

Region Habitat

BC wet edge of slough

BC wet edge of lake

BC in peat bog

NWT rare in moist black ground in
Carex meadow

BC somewhat calcareous swampy
lakeshore

AK herbaceous border of ponded
creek

AB creek bottom

Herbarium specimens identified as ITS-long: determined by sequencing or sizing assay to lack the
7 bp deletion in ITS2. Region abbreviations: BC British Columbia, MB Manitoba, MN Minnesota,
NC North Carolina, WA Washington, YT Yukon Territory.

Accession
no.

V226522

V67194

V96302

V97215

V233698

V122558

V228430

V195542

V240112

V227503

V237734

UTC00019311

MN71158

MN71175

Herb. Date

UBC 2007

UBC 1957

UBC 1950

UBC 1962

UBC 2007

UBC 1968

UBC 2006

UBC 1979

UBC 2010

UBC 2008

UBC 2007

USU 1935

MIN #1891

MIN 1891

Locality

Alaska Highway km 1016

Sage Creek, Flathead
Salmon Arm

Thompson-Nicola Regional District,
Tranquille

Greater Vancouver, Delta, Westham
Island

Avery County, Elk River at Heaton
Osoyoos, Haynes Point Provincial Park

Pencil Lake, Riding Mountain National
Park

Whitehorse

Vancouver Island, Duncan, Somenos
Marsh

Vancouver Island, Cumberland

Palouse River, Pullman

St Anthony Park, Ramsey

Ramsey

Region Habitat

YT apparently seeded along
highway

BC grassy meadow

BC not recorded

BC wet meadow

BC tidal shore (var. picta)

NC marsh

BC meadow beside wetland

MB road allowance, jet ski trail

YT sewage treatment facility

BC thick grassy marsh margin

BC roadside with introduced
grasses

WA shallow pools of drying
streambed

MN see Noyszewski et al. (2021)

MN see Noyszewski et al. (2021)

Report of two distinct ribotypes in ITS sequences of Phalaris arundinacea ... 5

Step description: DNA extraction, PCR and sequencing

Dried leaf material was ground to a slurry in liquid nitrogen and the DNA extracted using a
modified CTAB method (Doyle and Doyle 1987). Full length PCR (ITS1-5.8S-ITS2) was
performed using primers ITS-A (forward) (Blattner 1999) and ITS4 (reverse) (White et al.
1990) and PCR conditions 94°C/4 min, followed by 30 cycles of 94°C/30 sec, 50°C/1 min,
72°C/1 min and final extension of 72°C/10min. In cases of highly degraded DNA from older
herbarium specimens, ITS1 and ITS2 were amplified separately using primers ITS3P
(forward) (Moller and Cronk 1997) and the reverse complement ITS2P (reverse).
Bidirectional Sanger sequencing was performed by Eurofins (Kentucky, USA) and
sequences were checked using Sequencher version 4.8 (Gene Codes).

Sequence alignment and phylogenetic analysis

Sequences of 60 individuals were aligned manually using Sequencher and Se-Al
(Rambaut 2002). Subunit boundaries follow those determined for Oryza (Takaiwa et al.
1985, Yokota et al. 1989) as follows: 18S/ITS1 CATTG/TCGTG; ITS1/5.8S AAATC/
CACAC; 5.8S/ITS2 CACGC/CAAAA; ITS2/26S GGACC/GCGAC (an example of a full
Oryza sequence for location is GenBank accession MF029734). Eight putative hybrids
(between the different ribotypes) were excluded from the phylogenetic analysis due to
sequence superposition. We included one sample from GenBank (KF 753778) as the only
previously databased sequence with the ITS-short genotype. Phylogenetic analysis was
performed using three approaches: a Neighbour-joining (NJ) analysis with uncorrected (p)
distances and 1000 bootstrap replicates, a Maximum Parsimony (MP) analysis with
heuristic search (random addition of taxa and TBR branch swapping), both methods being
performed in PAUP* (Swofford 2003); and a Maximum Likelihood (ML) analysis using
RAXML (v. 8.2.4) with GTRCAT, 1000 rapid bootstraps and Gamma optimisation of tree
space run on the CIPRES Science Gateway (Miller et al. 2010, Stamatakis 2014). The MP
analysis also included a gap code matrix (for nine gaps: three in P. arundinacea and six in
outgroup taxa). Sequences are deposited in GenBank under accession numbers:
0Q740187-0Q740255.

Structural analysis of ITS2

Structural analyses were performed using the ITS2 database (Ankenbrand et al. 2015). We
used the Phalaris arundinacea |TS2 structure of GenBank accessions FJ821785 (MFE
-66.8 kcal/mol) in the ITS2 database for homology modelling (Wolf et al. 2005) of our
common variant (ITS-long) as it had a near identical sequence. As homology modelling of
the rare variant (ITS-short) fails on FJ821785, alternative templates for homology
modelling were investigated. Plausible configurations for ITS2-short were obtained using
Arctagrostis latifolia (EU792351) and Phalaris canariensis (FJ377670) as templates.

Capillary sizing assay

A primer was designed using the NCBI Primer-BLAST tool (Ye et al. 2012) ITS2AindelR: 5’-
GCAGCCATATCTTCGGC-3’ for use in conjunction with ITS primer ITS3P to allow an
accurate sizing assay on an ABI 3730 automated DNA Sequencer (Applied Biosystems).

6 Percy D, Cronk Q

The primer was combined with a M13 tail (5'-TGTAAAACGACGGCCAGT-3') on the
forward primer to facilitate fluorescent dye labelling and a further PIG tail (5°-GTTTCTT-3’)
on the reverse primer to promote terminal adenylation. We used a hot start touchdown
PCR protocol with 95°C/3 min, followed by 10 cycles of 94°C/30 sec, 65°C/30 sec (-1°C
per cycle, R 3°C/sec), 72°C/45 sec, followed by a further 30 cycles of 94°C/30 sec, 55°C/
30 sec, 72°C/45 sec and a final extension at 72°C/4 min. PCR products were loaded into
the capillary machine at 1:30 dilution and traces read using the programme Geneious 8.1.9
(Biomatters Ltd.). The PCR assay was designed to give products of 238 or 245 bp
depending on the presence of the 7 bp deletion.

A sequencing survey and phylogenetic analysis reveals intraspecific divergence in
ITS including a 7 bp deletion

Initial results of an ITS sequencing survey of Phalaris arundinacea from western Canada
revealed two distinctive sequences. One is full length with fixed heterozygosity
characteristic of polyploids; the other is shorter, with a 7 bp deletion in ITS2 and with no
fixed heterozygous base positions. The differences are summarised in Table 3. The tree
topologies recovered from the different phylogenetic approaches were nearly identical. The
matrix length was 603 bp (612 characters with gap coding) and the MP search recovered
two trees with length 117 (Cl: 0.93, Rl: 0.98); we present the strict consensus topology in
Fig. 2 showing majority rule consensus values as well as NJ and ML bootstrap support
values. The best ML model fit for the data (AIC) was GTR+G (-InL 1611.45). Use of
outgroups showed that the full length sequence (which we call ITS-long) was likely the
ancestral one and the deletion (ITS-short) is a putatively-derived character so far known
only from plants in north-western North America (Fig. 3). When compared with all available
world-wide sequences from GenBank (including Asia, Europe, North and South America),
only one sequence was found to have the ITS-short genotype (KF 753778) from Cook Inlet,
Alaska; all other GenBank samples are the ITS-long genotype and ITS-long sequences
found in North America are highly similar or identical to European genotypes. Tables 1, 2
show to which clade (ITS-long/-short) historical herbarium specimens can be assigned.

Table 3.

Molecular characteristics of the 7 bp deletion clade (ITS-short) in comparison to the full length
variant (ITS-long). Length variation in Phalaris arundinacea is caused by one 7 bp deletion and a 1
bp homopolymer indel, giving a combined length difference of 6 bp. The aligned sequence length
for 52 Phalaris arundinacea individuals using the ITS1-5.8S-ITS2 subunit boundaries following
Takaiwa et al. (1985) and Yokota et al. (1989) is 600 bp and, including six outgroup taxa (eight
individuals), it is 603 bp. Ambiguity codes (Y, R, S) are given for heterozygotes. Sites homozygous,
but polymorphic between different individuals, are given as C/T etc. Individuals that were
interpreted as putative hybrids are given in Suppl. material 1.

Feature ITS-long ITS-short Outgroups
No. of individuals 37 15 8
Total sequence length, ITS1-5.8S- 599 (no variation) 593 (no variation) 598-600

ITS2 (bp)

Report of two distinct ribotypes in ITS sequences of Phalaris arundinacea ...

Feature ITS-long ITS-short

ITS1 length 219 219

5.8S length 164 164

ITS2 length 216 210

Fixed heterozygosity in ITS1-long Y(30), Y(181), ¥(193), C(30), C(181), C(193),
(aligned position) S(208) C(208)

Fixed heterozygosity in 5.8S-long Y(345), Y(359) C(345), C(359)

(aligned position)

Fixed heterozygosity in ITS2-long Y(421), R(489), C(421), A/R(489),
(aligned position) Y(587) C(587)

Fixed SNPs between groups inITS1 —A(7), C(60), C(195) C7), T(60), Y/T(195)
(aligned position)

Fixed SNPs between groups inITS2 = 1(413), C(493), T(628) C(413), T/Y(493),C/
(aligned position) Y(528)

SSR in ITS2 (aligned position) C5(404-408) C5(404-409)

UTCO0019311 *USU 1935, WA
B5337 2010, USDA "PIBOsite75"
BRCO1 2021, BC

Cang-e1 modern, Europe

MN71175 *MIN 1891, MN
MN71158 *MIN $1891, MN

P. arundinacea
ITS-long

100
78/99

*UBC 1979, MB
*UBC 2007, YT
*UBC 2008, BC
*UBC 2006, BC
*UBC 2007, BC
*UBC 2007, BC
*UBC 2010, YT
V67194 *UBC 1957, BC
V96302 *UBC 1950, BC
DPQC10A 2021, AB

100/100

P. arundinacea

*UBC 1955, NWT
ITS-short

V162493 *UBC 1975, BC

*UBC 2014, BC

*UBC 1950, NWT

*UBC 1957, BC

*UBC 1957, BC

*UBC 1957, BC

V88503 *UBC 1958, BC

V119175 *ALA 1994, AK [KF753778]
V128867 *UBC 1970 P. caroliniana, LA
V106316 *UBC 1954 P. aquatica, OR
V196075 *UBC 1983 P. aquatica, OR
V106656 *UBC 1950 P. paradoxa, CA
V222762 *UBC 1992 P. coerulescens, Portugal
L12636 *UBC 2021 P. canariensis, BC
L12638 *UBC 2021 P. canariensis, BC
V195437 *UBC 1988 P. canariensis, BC

100/100

700/100
99/100 100

100/100

= 1 change

Figure 2. EESl

Outgroups
219-222
164
213-216

T/C(30), C(181), T/
C(193), C(208)

C(345), C/T(359)
C/T(421), G(489), C(587)
C(7), C/T(60), C(195)

T(413), C(493), T(528)

C?-6(404-409)

Phylogeny of 53 individuals of Phalaris arundinacea and five outgroup taxa, based on ITS
variation. Included are 60 individuals sampled for this study as well as the only GenBank
sample of P arundinacea found with the ITS-short sequence [KF753778]. Asterisks indicate
samples obtained from herbarium material. The tree is a strict consensus from the MP
analysis with Majority Rule consensus values above nodes and NJ/ML bootstrap support
values below nodes. Two clades can be seen: the deletion clade (ITS-short) and the full length
ITS clade (ITS-long). Sample details are given in Tables 1, 2 and Suppl. materials 1, 2, 3.

8 Percy D, Cronk Q

65

60

55

50

45
-160 -150 -140 -130 -120 -110 -100

Figure 3. EE)

Map of western Canada showing the locations of 51 genotyped samples of Phalaris
arundinacea. Dotted line indicates the Province of British Columbia. Red dots show the
locations of the “short” ribotypes (n = 13); blue “long” (n = 32) and orange putative hybrids (n =
6). Only three placeholder specimens are given for Elk Island National Park (arrowed; see
additional map Suppl. material 4). Sample details are given in Tables 1, 2 and Suppl. materials
ta 2 52

The 7 bp deletion alters the secondary structure of helix | of ITS2. The predicted secondary
structure of the common variant (ITS2-long) of Phalaris arundinacea |TS2 is the usual
eukaryotic four helix model (Fig. 4). Homology modelling of the structure of the ITS2-short
sequence against this structure fails, as helix |, which has the 7 bp deletion, is not a
suitable model. However, homology modelling with a related grass of similar ITS2
sequence suggests a plausible model for helix | despite the deletion (Fig. 4).

A PCR-based capillary sizing assay allows rapid detection of the 7 bp deletion clade

In order to genotype individuals without sequencing, we developed a primer that amplifies
a 238 vs. 245 bp amplicon (short enough to size accurately to a single base pair on a
capillary machine). ITS-long gave a clear peak at 245 bp and a complete absence of a
peak at 238 bp. Despite using a design to promote terminal adenylation (see Methods), if
there is a large amount of starting DNA, this peak was split, showing a peak or shoulder at
244 bp. However, in all cases, the fully adenylated peak was unambiguous and as strong
or stronger than the unadenylated peak. ITS-short samples gave a strong, unambiguous
peak at 238 bp. Product without terminal adenylation sometimes showed as a shoulder, but

Report of two distinct ribotypes in ITS sequences of Phalaris arundinacea ... 9

never a separate peak. ITS-short samples sometimes showed a small peak at 245 bp, but
the 238 peak was, in all cases, much stronger. A total of 68 individuals were sized with this
method, providing clade (ITS-long/-short/hybrid) affiliation for an additional 34 individuals.
Putative hybrids (10 individuals) were identified either by both sequencing and sizing assay
(seven individuals), sequence data only (one individual) and two specimens identified as
ITS-short in the length assay, but were determined as a putative hybrid with sequence data
(Suppl. material 1).

ITS-long

ITS-long

Figure 4. EES]

Secondary structure consequences of the deletion in ITS2. A) Predicted secondary structure
of Phalaris arundinacea ITS2, based on the common variant (ITS2-long). B) Detail of helix |;
DEL = the bases (GGGATGC) deleted in the ITS2-short variant; HR — C5 = 5 cytosine
homopolymer repeat; asterisk T — position of the T/C single nucleotide polymorphism (aligned
position 413). C) Possible alternative structure of helix | in the ITS2-short variant, based on
homology modelling using Arctagrostis latifolia as the template; the cytosine homopolymer
repeat is now C6 (6 cytosines); the arrow shows the position of the deleted sequence.

A survey of Elk Island National Park, Alberta, reveals presence of both ITS ribotypes

Using the molecular tools detailed above, we were able to conduct extensive sampling of
Elk Island National Park (EINP), Alberta. Phalaris arundinacea is extremely abundant at
EINP and the material in the Park tends to be strongly spreading-rhizomatous and
invasive. EINP is bisected into a northern and southern portion by the east-west highway
16. These portions have different management histories, with the northern portion
experiencing much greater public access and road development. We refer to these
portions as north EINP and south EINP. In all sampled localities of north EINP, ITS-long
was the only genotype detected (except a few possible hybrids at Tawayik Lake). In south
EINP the situation is very different. Of the 12 individuals genotyped from south EINP, five
were ITS-short (DPQC10A and DPQC11A-D).

10 Percy D, Cronk Q

Geographic coverage

Description: North-western North America

Taxonomic coverage

Description: Phalaris arundinacea, P. aquatica, P. canariensis, P. caroliniana,
P. coerulescens and P. paradoxa.

Usage licence

Usage licence: Creative Commons Public Domain Waiver (CC-Zero)

Data resources

Data package title: Specimen details for all 94 samples genotyped (86 Phalaris
arundinacea and eight outgroup taxa sampled).

Number of data sets: 1

Data set name: Specimen details for all 94 samples genotyped (86 Phalaris
arundinacea and eight outgroup taxa sampled).

Description: Suppl. material 3 contains specimen details for all 94 samples genotyped
(86 Phalaris arundinacea and eight outgroup taxa sampled).

Column label Column description

occurrence|ID Specimen Code identifier for the Occurrence.

basisOfRecord Specimen type as the specific nature of the data record.

eventDate Date of specimen collection.

eventRemarks Note of incomplete date information.

decimalLatitude The geographic latitude (in decimal degrees, using the spatial reference system given in

geodeticDatum) of the geographic centre of a Location.

decimalLongitude The geographic longitude (in decimal degrees, using the spatial reference system given in

geodeticDatum) of the geographic centre of a Location.

geodeticDatum The ellipsoid, geodetic datum or spatial reference system (SRS) upon which the geographic

coordinates given in decimalLatitude and decimalLongitude are based.
eventRemarks Ribotype of ITS sequence.
country The name of the country or major administrative unit in which the Location occurs.

locality The specific description of the place.

Report of two distinct ribotypes in ITS sequences of Phalaris arundinacea ... 11

verbatimLocality The original textual description of the place.

scientificName The full scientific name, with authorship.

identificationQualifier Qualifier on current identification.

taxonRank The taxonomic rank of the most specific name in the scientificName.

institutionCode The name (or acronym) in use by the Herbarium institution having custody of the object(s) or

information referred to in the record.

collectionCode The name, acronym, coden or initialism identifying the collection or dataset from which the

record was derived.

Additional information
Implications of two highly divergent intraspecific ribotypes

The making of a ribosome is a complex process: it involves multiple steps and over 200
biogenesis factors (Saez-Vasquez and Delseny 2019). In this process, ITS2 plays an
important role. The excision of ITS2 from the pre-ribosomal RNA is essential to generate
mature 25/26S and 5.8S and the secondary structure of ITS2 is important for this process
(Schultz et al. 2005). Embryophytes have four helices (numbered I-IV) arising from a
central ring. These helices require complementary base pairing to form (and be stable).
They are, therefore, generally quite conserved in sequence, with mutations only surviving if
they preserve the pairing energetics of the helix (Zhang et al. 2020). For this reason, it is
surprising to see an intraspecific seven base-pair deletion in helix |. In addition, this helix
carries an SNP and an extra cytosine in a cytosine repeat sequence. There is an
energetically plausible alternative structure for this helix, but it still represents a marked
change in helix pairing structure. Furthermore, there are seven SNPs in helix III (although
these do not markedly impact helix structure). Given this, it is evident that there are two
distinctive ITS2 ribotypes in north-western Canada, being distinguished by two indel
events, one with a major impact on helix nucleotide pairing and five SNPs.

The ITS-long sequence was highly similar or identical to sequences of known European
genotypes obtained from GenBank. In contrast, the ITS-short individuals are often from
non-agricultural and remote localities, (e.g. Yoho NP and Cook Inlet Lowlands of Alaska
and North West Territory) or from older herbarium specimens (e.g. a 1945 specimen from
Fort Saskatchewan, AB). These ITS-short genotypes are almost uniformly from riparian
and lacustrine habitats and never grassland. This genotype is currently unknown outside
north-western North America. Of historical and previously studied samples, the late 19!
century samples (1891), obtained from mid-western North America, Minnesota and
proposed as native genotypes in that region by Noyszewski et al. (2021), had the ITS-long
genotype in our study; a 1935 specimen from Pullman, Washington, proposed to be an
early European introduction by Kettenring et al. (2019), also had the ITS-long genotype;
and a modern (2010) specimen from remote northern BC (Kitimat), interpreted as native by
Jakubowski et al. (2014), but of mixed heritage by Kettenring et al. (2019), also had ITS-

12 Percy D, Cronk Q

long in our study. In summary therefore, across North America, the ITS-long genotype may
be present in both native and introduced RCG, whereas the ITS-short genotype appears to
be a localised variant in the Pacific northwest.

The existence of distinctive North American genotype(s) (e.g. Noyszewski et al. (2021))
suggests that RCG was widespread in North America prior to the massive seeding of
introduced agronomic genotypes in forage and revegetation seed mixes (Merigliano and
Lesica 1998). However, there is still much uncertainty and, in some cases, disagreement,
regarding the extent of distribution and location of present-day native RCG populations in
North America (Jakubowski et al. 2013). One potential use of our relatively easily scored
genetic marker would be to establish representation and association of the different
ribotypes in native populations. Preliminary observations of the growth forms of our
sampled RCG suggests that specimens with the ITS-short ribotype tended to be smaller,
less strongly rhizomatous and were not noted to be invasive. However, there is no
morphologically reliable method of distinguishing native from invasive RCG (Kettenring et
al. 2019). The main indicators are vigour of growth and rhizomatous spread, but the
usefulness of these indicators is uncertain and subject to environmental variation.
Presently, molecular markers will likely remain the primary means of making broad surveys
of RCG, to determine the geographical and ecological patterns of native persistence and to
identify cryptic invasions of RCG across North America and the potential signature of
intraspecific hybrids.

Acknowledgements

We thank Karin Kettenring, Karen Mock, Jim Walton (Utah State University) and Adam
Pidwerberski (Prince Albert National Park, SK) for material of RCG; Frank Lomer for
material of Phalaris canariensis; Timothy Whitfeld (Collections Manager) and staff of the
University of Minnesota Herbarium, Bell Museum (MIN) for loan of specimens; Spencer
Goyette and Linda Jennings (UBC Herbarium) for assistance; Hanna Schoenberg and
Pinette Robinson for assistance with work at Elk Island National Park (contract no:
085-5P426); Edward Sun (UBC) for assistance in the laboratory; and Neil Anderson
(University of Minnesota) for advice. We are grateful for a permit (no: 21-362) to collect at
Miquelon Lake issued by Alberta Environment and Sustainable Resource Development.
Lastly, we are grateful for reviews by Jeffrey Saarela, Neil Anderson and one annonymous
reviewer that helped improve the manuscript.

Author contributions

QCBC planned the research; DMP conducted the lab work; both authors collected and
analysed the data and both authors wrote the manuscript.

Report of two distinct ribotypes in ITS sequences of Phalaris arundinacea ... 13

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Report of two distinct ribotypes in ITS sequences of Phalaris arundinacea ... 15

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Supplementary materials

Suppl. material 1: Ten putative hybrids between ITS-long and ITS-short clades.
[doi |

Authors: Diana M. Percy, Quentin C. B. Cronk

Data type: occurrences

Brief description: Ten putative hybrids between ITS-long and ITS-short clades. Seven based on
both sequence and assay data, one based on sequence data only (marked “) and two samples
which appeared hybrid in sequence data, but ITS-short in sizing assay data (marked with *).
Region abbreviations: AB Alberta, BC British Columbia.

Download file (24.88 kb)

Suppl. material 2: Herbarium specimens used as outgroups. EE

Authors: Diana M. Percy, Quentin C. B. Cronk

Data type: occurrences

Brief description: Herbarium specimens used as outgroups.
Download file (24.08 kb)

Suppl. material 3: Specimen details for all 94 samples genotyped. EE

Authors: Diana M. Percy, Quentin C. B. Cronk

Data type: occurrences

Brief description: Specimen details for all 94 samples genotyped (86 Phalaris arundinacea and
eight outgroup taxa sampled).

Download file (15.38 kb)

Suppl. material 4: Map of Elk Island National Park with the locations of 38
genotyped samples marked. EI

Authors: Diana M. Percy, Quentin C. B. Cronk

Data type: occurrences

Brief description: Map of Elk Island National Park with the locations of 38 genotyped samples
marked. Red crosses show the locations of the “short” ribotypes (n = 4); blue crosses “long” (n =
29), and orange circles putative hybrids (n = 5).

Download file (45.26 kb)